INVESTIGATION Genome-Wide Transcriptional Dynamics in the Companion Bacterial Symbionts of the Glassy-Winged Sharpshooter (Cicadellidae: Homalodisca vitripennis) Reveal Differential Gene Expression in Bacteria Occupying Multiple Host Organs Gordon M. Bennett*,1 and Rebecca A. Chong† *Department of Plant and Environmental Protection, University of Hawaii at Manoa, Honolulu, Hawaii 96822 and †Department of Integrative Biology, University of Texas, Austin, Texas 78712 ABSTRACT The agricultural pest known as the glassy-winged sharpshooter (GWSS) or Homalodisca vitripennis KEYWORDS (Hemiptera: Cicadellidae) harbors two bacterial symbionts, “Candidatus Sulcia muelleri” and “Ca. Baumannia bacteriomes cicadellinicola,” which provide the 10 essential amino acids (EAAs) that are limited in the host plant-sap diet. mutualism Although they differ in origin and symbiotic age, both bacteria have experienced extensive genome degradation co-evolution resulting from their ancient restriction to specialized host organs (bacteriomes) that provide cellular support and genomics ensure vertical transmission. GWSS bacteriomes are of different origins and distinctly colored red and yellow. obligate While Sulcia occupies the yellow bacteriome, Baumannia inhabits both. Aside from genomic predictions, little is symbioses currently known about the cellular functions of these bacterial symbionts, particularly whether Baumannia in different bacteriomes perform different roles in the symbiosis. To address these questions, we conducted a replicated, strand-specific RNA-seq experiment to assay global gene expression patterns in Sulcia and Bauman- nia. Despite differences in genomic capabilities, the symbionts exhibit similar profiles of their most highly expressed genes, including those involved in nutrition synthesis and protein stability (chaperonins dnaK and groESL) that likely aid impaired proteins. Baumannia populations in separate bacteriomes differentially express genes enriched in essential nutrient synthesis, including EAAs (histidine and methionine) and B vitamins (biotin and thiamine). Patterns of differential gene expression further reveal complexity in methionine synthesis. Bau- mannia’s capability to differentially express genes is unusual, as ancient symbionts lose the capability to in- dependently regulate transcription. Combined with previous microscopy, our results suggest that the GWSS may rely on distinct Baumannia populations for essential nutrition and vertical transmission. Many insect groups have obligate associations with heritable microbes (e.g., leafhoppers, cicadas, spittlebugs) (Buchner 1965; Braendle et al. that enable novel ecological adaptations (Buchner 1965; Baumann 2005; 2003; Koga et al. 2013). However, in some host groups that have ac- Douglas 2015). In particular, plant sap-feeding insects (Order: Hemiptera) quired novel symbionts more recently, such as sharpshooter leafhoppers have ancient associations with bacteria that supplement their nutri- (Cicadellidae: Cicadellinae), bacteriaconsistentlyoccupymultiplebac- tionally imbalanced diets with the 10 essential amino acids (EAAs) teriomes (Buchner 1965; Moran et al. 2003, 2005b). As a result of ancient and vitamins (McCutcheon and Moran 2010; Bennett and Moran 2015; intracellular associations with host bacteriomes, tightly controlled verti- Sudakaran et al. 2017). These bacteria are generally transmitted verti- cal transmission, and small population sizes, symbiotic bacteria experi- cally and they are intracellularly restricted to specialized host organs ence dramatic gene loss and rapid rates of molecular evolution (Moran called bacteriomes (Buchner 1965). Distinct bacteriome organs typically 1996; Andersson and Kurland 1998; Wernegreen 2015). The impact contain a single bacterial species even when a host relies on more than of these forces on basic bacterial cell function remains poorly un- one complimentary symbiont as is common in the Auchenorrhyncha derstood, particularly in complex symbiotic systems where hosts rely Volume 7 | September 2017 | 3073 on multiple partners with different origins, levels of genome decay, response and nutrition synthesis genes (Wilcox et al. 2003; Moran and tissue associations. et al. 2005a; Stoll et al. 2009b). Here, our results show that symbioses To understand (a) how companion symbiont gene expression between GWSS, Sulcia, and Baumannia also show typical high gene patterns are affected by the symbiotic condition, and (b) whether expression of chaperonin and nutrition genes despite differences in symbionts infecting multiple tissues exhibit distinct cellular functions, age and origin, underscoring the shared impact of the symbiotic condi- we conducted replicated transcriptomic sequencing (RNA-seq) of bac- tion. Intriguingly, Baumannia is capable of differentially expressing genes teriomes from the glassy-winged sharpshooter leafhopper (GWSS), between bacteriomes, which includes those involved in essential nutrition Homalodisca vitripennis. GWSS relies on a complex symbiosis with synthesis. Combined with previous microscopy studies (Buchner 1965; two obligate bacteria for synthesis of the 10 EAAs (Wu et al. 2006; Kaiser 1980), these results suggest that the host may rely on separate McCutcheon et al. 2007). Like many other hosts in the Auchenorrhyn- Baumannia populations for distinct symbiotic functions. cha, GWSS retains the ancestral symbiont “Candidatus Sulcia muelleri” (Bacteroidetes;hereafterSulcia), that has allied with the suborder since MATERIALS AND METHODS it emerged .280 MYA (Moran et al. 2003, 2005b; Bennett and Moran 2013). Sulcia is perhaps one of the oldest known symbionts; it has a Fluorescence in situ hybridization microscopy small genome in GWSS (243 kb) and is responsible for the synthesis of GWSS specimens were collected from crape myrtle (Lagerstroemia eight EAAs (Moran et al. 2005b; Wu et al. 2006; McCutcheon et al. indica) at the University of Texas at Austin (UTA) in August of fl 2009a). In sharpshooters, the remaining EAAs are provided by “Ca. 2016 for uorescence in situ hybridization (FISH) symbiont localiza- Baumannia cicadellinicola” (Gammaproteobacteria; hereafter Bauman- tion experiments. FISH analyses were conducted on whole bacteriomes nia;Wuet al. 2006). Baumannia is derived from the replacement of the of three replicate individuals following previous approaches with more ancient leafhopper symbiont “Ca. Nasuia deltocephalinicola” GWSS that determined Baumannia and Sulcia cell morphology fi (Betaproteobacteria) in a common ancestor of sharpshooters .80 MYA (Moran et al. 2005b). Insect samples were initially xed overnight in ’ fl (Moran et al. 2003; Bennett and Moran 2013). In GWSS, Baumannia Carnoy s solution. In order to eliminate potential auto uorescence, – retains a relatively large genome for an insect intracellular symbiont tissues were bleached in 6% H202 80% EtOH for 2 wk (Koga et al. (686 kb) (Wu et al. 2006; Moran and Bennett 2014). It encodes many 2009). Following bleaching, bacteriomes were rinsed with 100% EtOH fl cellular capabilities lost in other ancient symbionts, including the potential and PBSTx, and hybridized overnight with the uorescent probes, – – to selectively regulate gene expression. CFB319 rhodamine and PRO319 Alexa488, which target Sulcia and The acquisition of Baumannia coincided with the evolution of a Baumannia, respectively (Moran et al. 2005b). DNA was counter- novel bacteriome organ, which has also been found to occur in recent stained with DAPI. Tissues were slide-mounted and visualized on a fl symbiont replacements in spittlebugs (Cercopoidea; Moran et al. 2003; Nikon Eclipse TE2000-U epi uorescence microscope. Bennett and Moran 2013; Koga et al. 2013). Sharpshooter bacteriomes generally comprise paired red and yellow tissues located along the Sample preparation and RNA sequencing lateral edges of the abdomen (Figure 1, A and B) (Moran et al. 2005b). Three insect replicate pools for dissection and RNA sequencing were WhiletheredbacteriomehousesonlyBaumannia, the yellow one con- collected separately from L. indica on the UTA campus between June tains Sulcia and Baumannia (Buchner 1965; Kaiser 1980; Moran et al. and August 2015. Insects were kept on L. indica for 48 hr in controlled 2003, 2005b). Baumannia’s presence in multiple host tissues, and its po- growth conditions (25°, 12 hr light/dark cycle) to mitigate potential tential metabolic and functional flexibility, raises the intriguing possibility environmental effects on gene expression. For each replicate, 14 mature that it may have multiple roles in the symbiosis. females were CO2-treated and whole bacteriomes immediately dissected One approach to better understand symbiont function is to profile in Buffer A (25 mM KCl, 35 mM Tris, 100 mM EDTA, and 250 mM their total gene expression patterns in the tissues that they occupy. These sucrose) and preserved in RNAlater (Ambion). Tissue pools were then data can provide functional insight into dependent microbial partners centrifuged at 4° for 15 min at 13,000 · g and RNAlater decanted. RNA that cannotbe cultured or easilyseparated.For example, geneexpression was extracted with TRIzol (Invitrogen) and residual DNA removed with studies in the Aphid–Buchnera model system and a handful of other DNA-free (Ambion). Total RNA was sent to the UTA Genomic Se- insect symbioses have shown that bacteria generally highly express quencing and Analysis Facility for sequencing. Ribosomal RNA was genes involved in essential nutrition and protein homeostasis (e.g., depleted using a Ribo-zero